摘要
目的:利用ITS2序列对市售火把花根药材及其混伪品进行DNA分子鉴定,以确保其临床用药的安全和有效。方法:对收集到的火把花根药材及其混伪品样品进行DNA提取,ITS2引物的PCR扩增与双向测序;采用CodonCode Aligner V 6.0.2软件对测序峰图进行校对拼接,经隐马尔夫模型的HMMer注释,并预测其二级结构;使用MEGA6.0计算遗传距离,构建NJ系统发育树。结果:火把花根药材及其混伪品的ITS2序列长度均在210~224 bp之间,其种间K2P遗传距离为0.012~0.690,火把花根与雷公藤、东北雷公藤、苦皮藤、南蛇藤及金樱根ITS2条形码变异位点分别为5、7、27、26和88个,其差异较为明显,经与GenBank数据库进行比对的结果表明,ITS2序列及其二级结构能准确区分火把花根药材及其混伪品。结论:DNA条形码ITS2序列可作为火把花根药材真伪鉴别的有效手段,也为后期火把花根片中成药的质量控制及品质评价提供了技术保障。
Objective:To conduct DNA molecular identification of Tripterygium hypoglaucum roots and the adulterants by using ITS2 sequence,so as to ensure its drug safety and the accuracy of clinical medication.Methods:The DNA extraction,PCR amplification and bidirectional sequencing of ITS2 primers were performed on the collected Tripterygium hypoglaucum roots and the adulterants.CodonCode Aligner V 6.0.2 software was used to collate and splicing the sequencing peak diagram,annotated by HMMer of Hidden Markov model,the secondary structure was predicted.MEGA6.0 was used to calculate genetic distance and construct NJ phylogenetic tree.Results:The length of ITS2 sequences of Tripterygium hypoglaucum roots and the adulterants ranged from 210 bp to 224 bp,and the interspecific K2P genetic distance ranged from 0.012 to 0.690.There were 5,7,27,26 and 88 mutation loci in the ITS2 barcodes of the roots of Tripterygium hypoglaucum and Tripterygium wilfordii,Tripterygium regelii,Celastrus angulatus,Celastrus orbiculatus,Rosa laevigata,their differences were obvious.Comparison with GenBank database showed that ITS2 sequence and its secondary structure could accurately distinguish the Tripterygium hypoglaucum roots medicinal materials from the adulterants.Conclusion:The ITS2 sequence of DNA barcode can be used as an effective method to identify the authenticity of Tripterygium hypoglaucum roots,and also provides technical guarantee for the quality control and quality evaluation of Chinese patent medicine of Tripterygium hypoglaucum roots tablets in the later stage.
作者
王江瑞
赵建权
蔡蓓蕾
王学政
欧洪
WANG Jiang-rui;ZHAO Jian-quan;CAI Bei-lei;WANG Xue-zheng;OU Hong(Sinomune Pharmaceutical Co.,Ltd.,Wuxi 214194,China;Pharmceutical Factory of Chongqing Academy of Chinese Materia Medica,Chongqing 400800,China;Chongqing Academy of Chinese Materia Medica,Chongqing 400065,China;Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China)
出处
《中药材》
CAS
北大核心
2022年第6期1333-1337,共5页
Journal of Chinese Medicinal Materials
基金
重庆市科技部“科技助力经济2020”重点专项(SQ2020YFF0417688)
关键词
火把花根
混伪品
ITS2序列
DNA分子鉴别
二级结构
Roots of Tripterygium hypoglaucum(Lévl.)Hutch.
Adulterants
ITS2 sequence
DNA Molecular identification
Secondary structures
作者简介
王江瑞(1990-),男,硕士,工程师,研究方向:中药学与药物化学,Tel:15310605574,E-mail:309361637@qq.com;通讯作者:欧洪,Tel:15928114716,E-mail:ouhong@cdutcm.edu.cn。
